Method of inducing apoptosis in cancer cells using an extract of Melothria indica Lou and a method for purification thereof

ABSTRACT

The invention includes a method of inducing apoptosis in cancer cells by administering an extract of Melothria indica Lou to the cancer cells. The invention also includes a method of using an extract of Melothria indica Lou to induce apoptosis in cancer cells by administering the extract to the cancer cells. The cancer cells can be leukemia cells and prostate cancer cells. Further included in the invention is a method for purifying the Melothria indica Lou into an extract used for inducing apoptosis in cancer cells.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates generally to cancer treatment and morespecifically, to cancer treatments which selectively induce apoptosis incancer cells.

[0003] 2. Description of Related Art

[0004] Cancer was generally treated with combinations of surgery,chemotherapy and radiation with varying degrees of success. One waycancer has been targeted for treatment is that cancer cells tend to berapidly dividing cells. Thus, current chemotherapeutic approaches targetrapidly dividing tumor cells. This approach is generally ineffectivewhen the cancer is dormant or slow growing. These types of treatmentsalso impact other, noncancerous cells that divide rapidly, causingharmful side effects.

[0005] These harmful side effects, however, are not present intreatments that induce apoptosis in the cancer cells. Apoptosis is alsocalled “programmed cell death” or “cell suicide.” (Krammer, et al.,“Apoptosis in the APO-1 System”, Apoptosis: The molecular Basis of CellDeath, pp. 87-99 Cold Spring Harbor Laboratory Press, 1991). In contrastto the cell death caused by cell injury,.apoptosis is an active processof gene-directed, cellular self-destruction. Apoptosis serves abiologically meaningful function. (Kerr, J. F. R. and J. Searle, J.Pathol. 107:41, 1971).

[0006] One of the examples of the biologically meaningful functions ofapoptosis is the morphogenesis of an embryo. (Michaelson, J., Biol. Rev.62:115, 1987). Just like creating a sculpture, clay is added, and clayis removed; organ formation (morphogenesis) of an embryo relies on cellgrowth (addition of clay) as well as cell death (removal of clay). As amatter of fact, apoptosis plays a key role in the human body from theearly stages of embryonic development through to the inevitable declineassociated with old age. (Wyllie, A. H., Int. Rev. Cytol. 68:251, 1980).

[0007] The normal function of the immune, gastrointestinal andhematopoietic system relies on the normal function of apoptosis. Whenthe normal function of apoptosis goes awry, the result can be any of thefollowing diseases: cancer, viral infections, auto-immunedisease/allergies, neurodegeneration or cardiovascular diseases.

[0008] The idea that cancer may be caused by insufficient apoptosisemerged only recently (Cope, F. 0. and Wille, J. J., “Apoptosis”: TheMolecular Basis of Cell Death, Cold Spring Harbor Laboratory Press, p.61, 1991). This idea opens a door for a new concept in cancertherapy—cancer cells may be killed by encouraging apoptosis. Apoptosismodulation, based on the processes present in normal development, is apotential mechanism for controlling the growth of tumor cells. Restoringapoptosis in tumor cells is an attractive approach because it programsthe cancer cells to commit suicide.

[0009] Thus, the cancer cells can be killed without killing the host.The success of this treatment, however, is dependent upon theavailability of drugs that can selectively induce apoptosis in tumorcells without affecting normal cells.

[0010] From the preceding descriptions, it is apparent that thecompositions and methods currently being used have significantdisadvantages. Thus, important aspects of the technology used in thefield of invention remain amenable to useful refinement.

SUMMARY OF THE INVENTION

[0011] The present invention introduces such refinement. In itspreferred embodiments, the present invention has several aspects orfacets that can be used independently, although they are preferablyemployed together to optimize their benefits.

[0012]Melothria indica Lou (Melothria) is a perennial berbaceous vinethat thrives on hillsides and in the woods and bushes. Melothria, whichemerged recently, is a popular herb in Taiwan, China and India and isbelieved to be beneficial in the treatment of sore throats, acuteconjunctivitis and inflammation.

[0013] We have discovered, however, that the Melothria extractselectively induces apoptosis in cancer cells.

[0014] Thus, the invention includes a method of inducing apoptosis incancer cells by administering an extract of Melothria indica Lou to thecancer cells, with the greatest apoptotic effects known at this timeoccurring in leukemia cells and prostate cancer cells. The inventionalso includes a method of using an extract of Melothria indica Lou toinduce apoptosis in cancer cells by administering the extract to thecancer cells.

[0015] The invention further includes a method for purifying Melothriaindica Lou, which comprises the following steps:

[0016] Extracting the Melothria indica Lou with a solvent to form anextract, concentrating the extract, applying the extract to a SCD100reverse phase chromatography column equilibrated with phosphate buffersaline, developing the SCD-100 column using an isocratic elution ofphosphate buffer saline for about forty (40) minutes with a flow rate ofabout 0.4 ml/min, collecting at least fraction eleven (11) from thecolumn, applying fraction eleven to a RPP-100 reverse-phasechromatography column equilibrated with 0.1% trifluoroacetic acid inwater, developing the column by a linear gradient using solvent A andsolvent B, solvent A being 0.1% trifluoroacetic acid in water andsolvent B being water/CH₃CN/0.1% trifluoroacetic acid in a ratio of49.5:49.5:1, generating a gradient using 100% solvent A and 0% solvent Bat time zero, then increasing solvent B from 0% to 100% after 20 mlelution volume, collecting at least fraction two from the column,applying fraction two to a AX-100 anion exchange chromatography columnequilibrated with solvent C, solvent C being 10% phosphate buffer salinein water, eluting the column with a solvent gradient including solvent Cand solvent D, solvent D being 100% phosphate buffer saline, the solventgradient is created starting with 100% solvent C and 0% of solvent D attime zero, then increasing solvent D from 0% to 100% after 20 ml elutionvolume, collecting at least fraction 12 from the column, applyingfraction twelve to a silica gel column equilibrated with a solvent E,solvent C being CH₃CN:ethanol:water at a ratio of 84.5:15:0.5,isocratically eluting the column with solvent C, collecting at leastfractions eight and nine.

[0017] All of the foregoing operational principles and advantages of thepresent invention will be more fully appreciated upon consideration ofthe following detailed description, with reference to the appendeddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows that the increase in the concentration of the extractof Melothria indica Lou increases the apoptotic effect in leukemiacells;

[0019]FIG. 2 shows that the increase in concentration of the extract ofMelothria indica Lou increases the apoptotic effect in prostate cancercells;

[0020]FIG. 3 shows the fractionation of the extract by SCD HPLCchromatography;

[0021]FIG. 4 shows the fractionation of the extract of Melothria indicaLou by RPP-100 reverse phase chromatography;

[0022]FIG. 5 shows the fractionation of the extract of Melothria indicaLou by anion exchange chromatography;

[0023]FIG. 6 shows the fractionation of the extract of Melothria indicaLou by silica gel absorption chromatography;

[0024]FIG. 7 shows the results of the partially purified extract ofMelothria indica Lou on mice injected with leukemia cells; and

[0025]FIG. 8 shows the results of the highly purified extract on miceinjected with prostate cancer cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] While the invention will be described in connection withpreferred embodiments, it will be understood that it is not intended tolimit the invention to those embodiments. On the contrary, it isintended to cover all alternatives, modifications, and equivalents asmay be included within the spirit and scope of the invention as definedby the appended claims.

[0027] In its preferred embodiment, the invention includes an extract ofMelothria indica Lou (“Melothria”) that selectively induces apoptosis incancer cells. When the extract of Melothria was administered to leukemiacells (HL-60, ATCC cell line CCL 240), the characteristics of apoptosiswere observed, both morphologically and biochemically, in approximatelyninety percent (90%) of the leukemia cells.

[0028] After the leukemia cells were incubated with the Melothria, DNAwas extracted from the leukemia cells and run on an agarose DNA Gel, andthe “DNA ladder” was observed. The DNA ladder is indicative of cellularapoptosis. To corroborate that the leukemia cells were undergoingapoptosis, they were incubated with the Melothria for one (1) hour andthen stained with Hoescht dye for one (1) hour.

[0029] Under fluorescent light, the nuclei of the leukemia cells showthe characteristics of apoptosis. The nuclei have condensed, and thenuclear condensation is accompanied by the fragmentation of the DNA.Both of these occurrences are the morphological characteristics of cellsundergoing apoptosis.

[0030] The Melothria extract was also administered to normal cells (CCD39 Lu, ATCC cell line CRL 1498). The Melothria did not induce apoptosisin the normal cells. None of the characteristics of apoptosis weredetected in the normal cells after they had been incubated with theMelothria for sixteen (16) hours. Thus, the Melothria extractselectively induced apoptosis in the leukemia cells and not the normalcells.

[0031] When the Melothria was administered to prostate cancer cells(LNCaP, ATCC cell line CRL 1740), the characteristics of apoptosis wereobserved. Although, the rate at which the Melothria induced apoptosiswas slower with the prostate cancer cells than the leukemia cells. Whenthe prostate cells were incubated with Melothria, the earliest time atwhich apoptosis was observed was after approximately five (5) hours ofincubation.

[0032] Melothria induces apoptosis in a dose dependent manner, as thedosage increases the percentage of cancer cells that undergo apoptosiscorrespondingly increases. As the concentration of the extractincreases, the percentage of cancer cells undergoing apoptosisincreases. (See FIGS. 1 and 2).

PURIFICATION

[0033] Purification of the Melothria extract was performed to find theactive component in the extract. To partially purify the Melothria,fifty milliliters (50 ml) of hot water was added to ten grams (10 grams)of the fruit of Melothria indica Lou. The Melothria fruit and hot watersteeped for about ten (10) minutes. The solution was concentrated intoone milliliter (1 ml) using a SpeedVac. The concentrated solution wasthen passed through a Synchropak SCD-100 column. The column wasequilibrated with phosphate buffer saline (PBS) and then developed by anisocratic elution of PBS for forty (40) minutes with a flow rate of 0.4ml/min.

[0034] Each of the resulting fractions, one through twenty-four, wascollected and then tested on the leukemia cells. Fraction 11 inducedapoptosis in the leukemia cells. Fraction 11 was then further purifiedusing a RPP-100 reverse-phase column (Micra Scientific, Inc. Northbrook,Ill.). (See FIG. 3).

[0035] One milliliter (1 ml) of the concentrated active componentpresent in fraction 11 was applied to the RPP-100 equilibrated with 0.1%Trifluoroacetic acid (TFA) in water. The column was developed using alinear gradient created by solvent A and solvent B. Solvent A is 0.1%TFA in water and solvent B is H_(2 O:CH) ₃CN:0.1% TFA having a ratio of49.5:49.5:1. The gradient was generated by applying 100% solvent A and0% solvent B at time zero (0), followed by increasing solvent B from 0%to 100% after 20 ml elution volume. Fraction 2 of the elution profileinduces apoptosis in the leukemia cells. (See FIG. 4).

[0036] To further purify the extract, fraction 2 was applied to anAX-100 anion exchange chromatography column (Micra Scientific Inc.,Northbrook, Ill.). The AX-100 was equilibrated with 1:10 H₂O dilution ofPBS, solvent C. The column was then eluted with a gradient created by10% PBS in water, solvent C, and 100% PBS, solvent D. The gradient wascreated using 100% solvent C and 0% solvent D at time zero (0) followedby increasing solvent D from 0% to 100% after 20 ml elution volume.Fraction 12 of the elution profile induced apoptosis in prostate cancercells (LNCaP) and the leukemia cells (HL-60). (See FIG. 5).

[0037] To purify the extract even further, fraction 12 was applied ontoa silica gel column (Partisil 5, Whatman) which was equilibrated withCH₃CN:Ethanol:H₂O having a ratio of 84.5:15:0.5, solvent E. The columnwas isocratically eluted with solvent E. Fraction 8 and fraction 9 ofthe elution profile induced apoptosis in prostate cancer cells andleukemia cells. Fractions 8 and 9 include the active component of theextract in a highly purified form. (See FIG. 6).

TESTING AND RESULTS OF PARTIALLY PURIFIED EXTRACT

[0038] A test was run using extract that had been partially purified bythe SCD-100 column. The partially purified extract of Mel othria indicaLou increased the life span of leukemia-bearing mice. Forty (40) DBA/2female mice (17-20 grams; Simonsen Laboratories, Inc., Gilroy, Calif.)were inoculated with the tumor cell line P388D1 (ATCC Cell Line CCL 46).The tumor cells were harvested and diluted with saline. Then, the tumorcells were checked for viability using the trypan-blue stainingtechnique. Each mouse was injected intraperitoneally with one milliontumor cells. The mice were then segregated into four groups of ten mice.The mice were housed in shoe box cages and kept on a standard diet withwater ad libitum.

[0039] Fraction 11 of the partially purified Melothria extract describedabove, was intraperitoneally injected into three of the groups. Each ofthe three groups was injected with different dosages of the extract. Thefourth group was injected with saline.

ILS=100×median life span of treated mice−median life span of controlmice life span of control mice

[0040] Referring to FIG. 7, Group I received a 0.002 ml dose of fraction11, which increased the life span of the mice by twenty-five percent(25%). Group II received a 0.02 ml dose of fraction 11, which increasedthe life span of the mice by forty-five percent (45%). Group IIIreceived a 0.2 ml dose of fraction 11, which increased the life span ofthe mice by one hundred fifty percent (150%). Group IV received 0.5 mlof saline solution and provides the life span from which the increasedlife span is determined. The induction of apoptosis is dose dependent,as the dose of the fraction 11 material is increased, the life span ofthe mice also increases. (See FIG. 7).

TESTING AND RESULTS OF HIGHLY PURIFIED EXTRACT

[0041] Highly purified extract was used in an in vivo study using micewith prostate cancer. Referring to FIG. 8, on day one, NIH nude adultmale mice (TACH:NA (S)-NuFDF Homozygous males 3-4 weeks old) wereinoculated with human prostate adenocarcinoma tumor cells (PC-3, ATCCCell Line CRL 1435). On day two (2), Group I, the control group,received 0.2 ml of saline injected intraperitoneally for five (5) days.Group II received 0.01 ml of the extract, Group III received 0.1 ml andGroup IV received 0.2 ml of the extract for five days.

[0042] The mice weighed an average of 22 g at the start of theexperiment, and 30 g at the end of the experiment six (6) weeks later.After six weeks elapsed, the mice were examined, and the tumors wereweighed. No tumors developed on the mice given the extract, groups II,III, and IV. Each mouse in the control group, however, developed tumors.Thus, the purified extract completely inhibited the formation of tumorsin the mice. (See FIG. 8).

I claim:
 1. A method of inducing apoptosis in cancer cells byadministering an extract of Melothria indica Lou to the cancer cells. 2.The method of claim 1 wherein the cancer cells are leukemia cells. 3.The method of claim 1 wherein the cancer cells are prostate cancercells.
 4. A method of using an extract of Melothria indica Lou to induceapoptosis in cancer cells by administering the extract to the cancercells.
 5. The method of claim 4 wherein the cancer cells are leukemiacells.
 6. The method of claim 4 wherein the cancer cells are prostatecancer cells.
 7. A method for purifying Melothria indica Lou comprisingthe steps of: extracting the Melothria indica Lou with a solvent to forman extract; concentrating the extract; applying the extract to a SCD-100reverse phase chromatography column equilibrated with phosphate buffersaline; developing the SCD-100 column using an isocratic elution ofphosphate buffer saline for about forty (40) minutes with a flow rate ofabout 0.4 ml/min; collecting at least fraction eleven (11) from thecolumn; applying fraction eleven to a RPP-100 reverse-phasechromatography column equilibrated with 0.1% trifluoroacetic acid inwater; developing the column by a linear gradient using solvent A andsolvent B, solvent A being 0.1% trifluoroacetic acid in water andsolvent B being water/CH₃CN/0.1% trifluoroacetic acid in a ratio of49.5:49.5:1; generating a gradient using 100% solvent A and 0% solvent Bat time zero, then increasing solvent B from 0% to 100% after 20 mlelution volume; collecting at least fraction two from the column;applying fraction two to a AX-100 anion exchange chromatography columnequilibrated with solvent C, solvent C being 10% phosphate buffer salinein water; eluting the column with a solvent gradient including solvent Cand solvent D, solvent D being 100% phosphate buffer saline, the solventgradient is created starting with 100% solvent C and 0% of solvent D attime zero, then increasing solvent D from 0% to 100% after 20 ml elutionvolume; collecting at least fraction 12 from the column; applyingfraction twelve to a silica gel column equilibrated with a solvent E,solvent C being CH3CN:ethanol:water at a ratio of 84.5:15:0.5;isocratically eluting the column with solvent C; collecting at leastfractions eight and nine.